Common Acute Lymphoblastic Leukemia Research Articles

Abstract 2462: MondoA mediates in vivo aggressiveness of common ALL and may serve as a T-cell immunotherapy target

Abstract Oncogene addiction provides ideal targets for immunotherapy. We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Here we report on the expression of MondoA in common acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Given the non-accessibility of transcription factors by drugs or chimeric antigen receptor transgenic T cells (CARs), we tested the targetability of MondoA by allo-restricted, peptide specific T cells. Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA (shRNA). Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. MondoA specific T cells were generated by priming of donor HLAA0201 negative (A2-) T-cells with A2+ dendritic cells bearing MondoA peptides, multimer-based sorting and subcloning of A2-CD8+ T-cells. For priming of T cells, five MondoA peptides were chosen by SYMPEITHI, BIMAS and NetCTL1.2. analyses. Peptide 428 stabilized best A2 expression on TAP-deficient T2 cells. Specificity and functionality of T cell clones were tested by ELISpot interferon gamma (IFg) and granzyme B assays with six MondoA+ leukemia lines (A2+, A2-). Off target effects of MondoA specific T-cell clones were assessed by IFg reactivity against the MondoA expressing A2+ NALM6 cell line vs. A2+ and A2- EBV immortalized lymphoblastoid cell lines from six donors. Peptide homology was assessed with BLAST algorithms in SWISSPROT. We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in treated mice after MondoA KD. Further microarray analysis revealed an induction of aerobic glycolysis switch genes and hypoxia-response by MondoA. Consequently, HIF1A stabilization required MondoA expression and tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. Therapeutically, MondoA-derived peptide antigens and A2+ cALL lines were successfully recognized and killed by specific, allo-restricted CD8+ T cells. In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response. Moreover, we identified MondoA as a promising target for immunotherapy of cALL. Citation Format: Alexandra Sipol, Thomas G. P. Grunewald, Juliane Schmaeh, David Schirmer, Monique L. den Boer, Rebeca Alba Rubío, Michaela Baldauf, Caroline Wernicke, Hans-Jochem Kolb, Martin Horstmann, Gunnar Cario, Guünther Richter, Stefan Burdach. MondoA mediates in vivo aggressiveness of common ALL and may serve as a T-cell immunotherapy target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2462.

Plasma Neprilysin Concentrations: A New Prognostic Marker in Heart Failure?

Neprilysin, also known as neutral endopeptidase (NEP), endopeptidase 24.11, CD10, enkephalinase, and common acute lymphoblastic leukemia antigen (CALLA), is a membrane-bound metalloendopeptidase (EC 3.4.24.11) that has recently sprung to renewed prominence as a target in cardiovascular therapeutics. Numbered among the many substrates to NEP are multiple vasoactive peptides, including the cardiac natriuretic peptides, with important roles in regulating pressure and volume status in health and disease. In the 1990s, NEP inhibitors were combined with angiotensin converting enzyme inhibitor (ACEI) therapy with the hope of adding the expected multifaceted benefits of enhanced plasma and tissue natriuretic peptide levels to the proven benefits of ACEI-related suppression of adverse renin-angiotensin-aldosterone system activation. After initial promise from trials in hypertension and heart failure, drug development was halted due to an unacceptable incidence of angioneurotic edema. The current renewed interest in NEP reflects the recent success of a new combination, that of inhibition of NEP plus angiotensin II type 1 receptor blockade. The PARADIGM trial tested the NEP inhibitor sacubitril combined with the angiotensin receptor blocker valsartan (LCZ696) in chronic heart failure with reduced ejection fraction. The new treatment was significantly beneficial with respect to all important clinical endpoints and was associated with no excess of important adverse effects and in fact caused less renal failure and hyperkalemia than established evidence-based treatment with the ACEI enalapril. The results point to this strategy being the greatest advance in the pharmacotherapy of chronic heart failure in the last 20 years. In addition to its widespread tissue-based, membrane-bound form, NEP exists in a circulating nonbound form that retains catalytic activity. In the article published in Revista Espanola de Cardiologia, Bayes-Genis et al build on previous work to confirm their original finding that plasma NEP concentrations have

Increased μ-Calpain Activity in Blasts of Common B-Precursor Childhood Acute Lymphoblastic Leukemia Correlates with Their Lower Susceptibility to Apoptosis.

Childhood acute lymphoblastic leukemia (ALL) blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS) composed of cytoplasmic proteases—calpains—performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor—calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher μ-calpain (CAPN1) gene transcription, protein amounts and activity (but not those of m-calpain), with calpastatin amount and transcription of its gene (CAST) greatly varying were observed in CD19+ ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk) displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL.

In vitro study of BRD4 inhibitor GSK525762A against primary adult common B-cell acute lymphoblastic leukemia cells in vitro

目的探讨4型含Bromo结构域蛋白（BRD4）拮抗剂对普通型急性B淋巴细胞白血病（B-ALL）患者原代白血病细胞的作用及可能机制。方法采用流式细胞术分选14例普通型B-ALL患者（Ph+ B-ALL 4例，Ph−B-ALL 10例）白血病细胞，在模拟骨髓微环境条件下进行短期培养；给予BRD4拮抗剂GSK525762A处理后，采用CCK-8法检测细胞增殖抑制率，用AnnexinⅤ/7-AAD法检测细胞凋亡率，荧光定量PCR法检测c-MYC、CDK6、Bcl-2 mRNA表达水平，Western blot法检测c-MYC、CDK6、Bcl-2蛋白的表达。结果GSK525762A对14例普通型B-ALL患者原代白血病细胞均有抑制增殖作用，且呈剂量依赖性，其中位IC50值为256.25（90.64~1 378.39）nmol/L。500、1 000、2 500 nmol/L GSK525762A作用后中位细胞凋亡率分别为45.17%（9.38%~70.91%）、66.02%（24.36%~96.34%）、89.29%（39.29%~99.37%）。GSK525762A对Ph+与Ph−B-ALL细胞具有类似的作用，但对Ph+ B-ALL细胞抑制增殖和诱导凋亡作用均弱于Ph−B-ALL细胞。1 000 nmol/L GSK525762A处理白血病细胞24、48 h后，c-MYC、CDK6和Bcl-2 mRNA表达水平下降，其中对Ph+和Ph−B-ALL细胞c-MYC、CDK6 mRNA的下调作用差异无统计学意义，而对Ph+ B-ALL细胞Bcl-2 mRNA表达水平的下调弱于对Ph−B-ALL细胞；GSK525762A处理后白血病细胞c-MYC、CDK6、Bcl-2蛋白表达水平均下调。结论GSK525762A可抑制普通型B-ALL原代细胞的增殖，并促进其凋亡，且对Ph+ ALL细胞在体外具有一定作用。该作用可能通过下调c-MYC、CDK6和Bcl-2而实现。

A label-free immunosensor for detecting common acute lymphoblastic leukemia antigen (CD10) based on gold nanoparticles by quartz crystal microbalance

Rapid and sensitive detection of the common acute lymphoblastic leukemia antigen (CD10) is crucial for diagnosing and prognosing hematopoietic tumors and several carcinomas. In the present study, a label-free immunosensor for detecting CD10 by Quartz Crystal Microbalance (QCM) was developed. The detection system has a good selectivity based on the biospecific interactions between the CD10 antigen and antibody, and the sensitivity of the immunosensor was further improved for Au NPs not only carried more antibodies but also acted as mass enhancers. Experimental results confirmed that this developed method could be conveniently used for the detection of CD10 without labeling, and CD10 could be measured quantitatively in the concentration range from 1.0×10−8 to 1.0×10−11M.

Regenerating blasts masquerading as relapse in a patient with ALL following G-CSF therapy.

TO THE EDITOR: Granulocyte colony-stimulating factor (G-CSF)-associated blastocytosis with a lack of an associated left shift poses a challenge for accurate assessment of the underlying disease [1]. In particular, blastocytosis after chemotherapy for acute leukemia may mimic a recurrence of underlying leukemia. Only a few cases of myeloblastosis in AML or ALL or of leukoerythroblastosis in AML after G-CSF therapy have been described anecdotally in the literature [2,3,4]. Herein, we report a case of myeloblastosis in ALL within 2 days of G-CSF administration masquerading as early relapse, causing a diagnostic dilemma. This was compounded by a striking lack of telltale evidence that G-CSF treatment was effective, such as increased granulation and a left shift. A 25-year-old man diagnosed with common acute lymphoblastic leukemia antigen [CALLA] positive, precursor B-cell ALL (Fig. 1A) completed his first phase of induction chemotherapy with methotrexate, cyclophosphamide, and cytarabine. His day 36 bone marrow showed 2.8% blasts with positive minimal residual disease (MRD) (0.046%). To accelerate recovery from persistent neutropenia, he was treated with G-CSF; an adequate response was noted, with a total leukocyte count (TLC) of 2.9×109/L. On day 70, he completed his second phase of induction chemotherapy and was started on G-GSF therapy for severe neutropenia (TLC: 0.76×109/L). On day 73, his peripheral blood smear showed marked leukopenia with a predominance of lymphocytes and 2% blasts without any left shift or increased granulations. This lack of prototypical G-CSF response along with previous MRD positivity and presence of 2% blasts led to a suspicion of relapse, and bone marrow examination was advised. Marrow aspirate showed hypocellular marrow with 14% blasts, and focal areas showed up to 25% blasts (Fig. 1B), along with a lack of a maturing myeloid component, which suggested relapse. Trephine biopsy, in contrast, showed hypocellularity with only a few scattered CD34 positive blasts. This necessitated flow cytometric evaluation, where the blasts showed negativity for B lymphoblastic markers and expressed myeloid markers, including CD13, CD33, and CD34, suggesting that they were regenerating myeloblasts (Fig. 1C-F). No further therapy was administered and the patient was kept on close hematological follow-up. The counts recovered after 4 days, with the typical responses of a left shift and increased granulation. Follow-up marrow examination after complete recovery of blood counts, 10 days after discontinuing G-CSF therapy, revealed cellular marrow elements with 4% blasts (regenerating) and negative MRD (<0.01%). Fig. 1 (A) Diagnostic blasts with a high nucleocytoplasmic ratio and scant agranular cytoplasm (Giemsa stain, ×1,000). (B) Regenerating blasts are larger with moderate granular cytoplasm (Giemsa stain, ×1,000). (C-F) Immunophenotyping showing ... Post-G-CSF blastocytosis is a problematic and confusing issue that is difficult to differentiate from disease recurrence and hemopoietic recovery. In our case, there were regenerating blasts (>20%) in the bone marrow, with an absence of telltale G-CSF-induced changes, such as presence of immature granulocytes, monocytosis, or prominent granulations on peripheral smear. To conclude, the regenerating blast percentage may increase after G-CSF therapy to >20%, without concomitant increases in other mature myeloid precursors. The lag time before complete response to G-CSF therapy may be as long as 10 days, as seen in our case, posing potential problems in management and a challenge in therapeutic decisions. A careful morphological assessment of the blasts at the time of diagnosis, aided by awareness of these G-CSF-associated aberrant patterns and flow cytometric characterization can help guide management decisions.

IKZF1 Status As an Independent Prognostic Marker in Adult Common B-Cell Acute Lymphoblastic Leukemia

Previous studies have suggested that IKZF1 gene deletions are associated with an adverse prognosis in acute lymphoblastic leukemia (ALL). However, the prognostic value of IKZF1 deletions in adult common B-cell ALL (Com-B ALL) has not been well-defined, especially in the context of different post-remission therapies. The objectives of this study were to evaluate the prognostic role of IKZF1 deletions in adult Com-B ALL.Overall, 162 untreated adult Com-B ALL patients were recruited between April 2006 and April 2013. Patients received one or two cycles of induction therapy and, if in remission, were allowed to select either ongoing systemic chemotherapy or hematopoietic stem cell transplantation (HSCT). BCR-ABL positive patients were additionally treated with tyrosine kinase inhibitors (TKIs). Deletions in the IKZF1 gene were detected using multiplex RQ-PCR, multiplex fluorescent PCR, multiplex ligation-dependent probe amplification (MLPA) and sequence analysis. High-risk (HR) was defined as having any of the following factors: central nervous system involvement at diagnosis; age ¡Ý50 years; WBC count ¡Ý30x109/L; hypodiploidy; mixed lineage leukemia (MLL) gene rearrangements; BCR-ABL rearrangements; t(1;19) translocations; or a complicated karyotype (¡Ý5 chromosomal abnormalities); failure to achieve remission after two cycles of induction therapy. The study end points included relapse-free survival (RFS), disease-free survival (DFS) and overall survival (OS), which was analyzed using the Kaplan-Meier method. The cumulative incidence of relapse (CIR) was estimated, adjusting for competing risks of death, and compared by Gray’s test.IKZF1 deletions were detected in 79 (48.8%) out of 162 adult Com-B ALL patients. Patients with an IKZF1 deletion (n=79) had a significantly inferior prognosis than those with wild-type IKZF1 (n=83) (Fig.1). The prognosis of patients with IKZF1 deletions with or without the BCR-ABL rearrangements was significantly inferior compared to those of patients with neither IKZF1 deletions nor BCR-ABL rearrangements. The power of IKZF1 deletion status as a prognostic factor remained in both non-HR group (5-yr OS: 52.5%¡À13.1% for IKZF1 deletions (n=25) vs. 72.2%¡À8.8% for wild-type IKZF1 (n=42), P=0.048) and HR group (5-yr OS: 36.7%¡À8.2% for IKZF1 deletions (n=54) vs. 69.3%¡À8.3% for wild-type IKZF1 (n=41), P=0.015). Among patients who received chemotherapy as post-remission therapy (n=52), IKZF1 deletions were associated with an unfavorable OS (P=0.003). In this group, the median OS time for IKZF1-deletion patients (n=28) was 10.0¡À1.0 months, while the median OS time for IKZF1-wild-type carriers (n=24) was not achieved. Among those that received HSCT as post-remission therapy (n=94), IKZF1 status did not affect the prognosis.For patients with IKZF1 deletions, HSCT (n=40) was significantly superior to chemotherapy (n=28) as a post-remission therapy. In the wild-type IKZF1 HR group, patients that received HSCT (n=24) achieved a longer DFS and RFS while the OS did not significantly differ from that of patients who received chemotherapy alone (n=12). In the wild-type IKZF1 non-HR group, there was no significant difference between patients that received HSCT (n=30) and those who received chemotherapy (n=12) as a post-remission therapy. Comparing survival curves of HSCT to chemotherapy are shown in Fig.2. Multivariate analysis confirmed the negative impact of IKZF1 deletions on OS (RR 2.3; 95% CI, 1.3-4.3; P=0.007), DFS (RR 1.8; 95% CI, 1.1-3.0; P=0.029), RFS (RR 2.1; 95% CI, 1.3-3.9; P=0.018) and favorable impact of HSCT on OS (RR 0.2; 95% CI, 1.0-0.5; P=0.000), DFS (RR 0.2; 95% CI, 0.1-0.4; P=0.000), RFS (RR 0.2; 95% CI, 0.1-0.4; P=0.000).These results indicated that IKZF1 status served as an independent negative prognostic marker in adult Com-B ALL and surpassed conventional HR features in the era of TKIs. Patients may benefit from therapy stratified by IKZF1 deletions and conventional HR features. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Overexpression of the Metabolic Stress Sensor and Proglycolytic Transcription Factor ΜLΧΙΡ Mediates Malignancy of Common Acute Lymphoblastic Leukemia in Vivo and Is Targetable By Allorestricted Peptide Specific T Cells

Oncogene addiction provides ideal targets for successful immunotherapy. MLXIP (MAX like protein X interacting protein, also known as MondoA) is a metabolic stress sensor and a proglycolytic transcription factor potentially involved in metabolic addiction features of leukemia, the Warburg effect and anoikis. MLXIP dimerizes with MLX within the MYC interactome and promotes longevity in C. elegans (Johnson et al. 2014). The MYC interactome comprises the MYC/MAX/MAD/MLX/MLXIPtranscriptionfactornetwork: Its key players MYC, MAD and MLXIP differentially mediate proliferation, differentiation or metabolism by heterodimerization with MAX or MLX: While MAX is available for partnering with MYC vs. MAD, MLX may partner with MAD or MLXIP alternatively. In contrast to the perception of MLXIP as a tumor suppressor gene in some solid tumors such as breast and colon cancer (O’Shea and Ayer, 2013), we have extended our previous notion of up regulation of MLXIP in B cell acute lymphoblastic leukemia(ALL, Burdach & Richter 2007, Haferlach et al. 2010) by demonstrating that MLXIP is highly overexpressed and induces stemness, proliferation and B cell receptor signaling pathway signatures in common ALL (cALL, Wernicke et al. 2012). Here we report on the role of MLXIP in malignancy of cALL in vivo. Given the non accessibility of transcription factors by chimeric antigen receptor transgenic T cells (CARs) and the superiority of allorestricted T cells in T cell receptor (TCR) based immunotherapy of leukemia (Burdach & Kolb 2013), we also tested the targetability of MLXIP by allorestricted peptide specific T cells to ultimately generate MLXIP specific allorestricted TCR transgenic T cells (ATRs, instead of CARs).Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were retrovirally transduced with MLXIP short hairpin RNA (shRNA). Upon successful MLXIP knock down (kd), kd and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. MLXIP specific T cells were generated by priming of donor HLAA0201 negative (A2-) T-cells with A2+ dendritic cells bearing MLXIP peptides, multimer-based sorting and subcloning of A2-CD8+ T-cells. For priming of T cells, five MLXIP peptides were chosen by SYMPEITHI, BIMAS and NetCTL1.2. analyses. Peptide 428 stabilized best A2 expression on TAP-deficient T2 cells. Specificity and functionality of T cell clones were tested by ELISpot interferon g (IFg) and granzyme B assays with six MLXIP+ leukemia lines (A2+, A2-). Off target effects of MLXIP specific T-cell clones were assessed by IFg reactivity against the MLXIP expressing A2+ NALM6 cell line vs. A2+ and A2- EBV immortalized lymphoblastoid cell lines from six donors. Peptide homology was assessed with BLAST algorithms in SWISSPROT.We found MLXIP to be overexpressed in both ALL and AML, while it was low in solid tumors including breast and colon cancer. MLXIP kd in the cALL cell line NALM-6 reduced the transcript by 80%. Importantly, in vivo MLXIP maintained 90-99% of CD10+ leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized by MLXIP kd: While signs of leukemia engraftment were observed in all mice, the differences of CD10+ blasts in blood, marrow and spleens in the control vs. the MLXIP kd group were highly significant (p=0.008). The decrease of leukemic splenomegaly after MLXIP kd was impressive. Median spleen weight was 0.22g vs. 0.08g in the control vs. the kd group (Figure 1: The upper spleen displays the leukemic infiltration in control mice. The two lower spleens are from mice injected with MLXIP kd NALM6 cells).MLXIP peptide 428 specific T cell clones successfully recognized and killed A2 positive MLXIP expressing NALM6 and 697 cALL lines. In contrast, MLXIP specific T cell clones did not release IFg and granzyme B under stimulation with A2- REH, RS4;11 cALL lines. We identified the nucleotide sequence of alpha and beta variable chains of MLXIP specific A2 restricted TCR to finally obtain MLXIP specific ATRs. Peptide dependent and independent off target alloreactivity was very low compared to reactivity against NALM-6 cALL.In conclusion, these findings demonstrate that MLXIP maintains leukemic burden and malignancy of cALL in vivo. Moreover, we identified MLXIP as a promising target for immunotherapy of cALL and potentially other MLXIP expressing leukemias, including AML. [Display omitted] DisclosuresHaferlach:MLL: Equity Ownership. Burdach:PDLI: Equity Ownership.

Bone Marrow Microbiome: Metagenomic Comparsion of Childhood Acute Lymphoblastic and Acute Myeloid Leukamias

Purpose: To describe and compare the pre-treatment bone marrow metagenomes of childhood acute lymphoblastic leukemia (ALL) and childhood acute myeloid leukemia (AML).Background: Leukemia is the most common cancer in children, comprised mainly of ALL (~80%) followed by AML (~17%). The role of infections and their timing in the etiology of pre B-cell ALL’s (but not AML’s) is supported by a considerable body of epidemiologic evidence developed in the past 20 years (Gilham, Peto et al. 2005, Ma, Urayama et al. 2009). There are two main infection related hypotheses. Greaves postulates that delayed infection, akin to the hygiene hypothesis, increases risk of leukemia (Greaves and Alexander 1993, Greaves 1997). The second hypothesis set forth by Kinlen is not mutually exclusive and postulates the role of a transforming yet common virus where introduction of the infectious agent to previously naive communities trigger leukemia (Kinlen 1995). While many viral agents have been investigated (reviewed in (O’Connor and Boneva 2007)) no definitive infectious agent has been identified but, until recently, broad surveys of the microbiome were not possible. Recent developments in next generation sequencing and bioinformatics now allow for unbiased investigation into the totality of organisms that inhabit the human body.Methods: Pre-treatment bone marrow specimens were collected in pre B-cell ALL and AML cases participating in the California Childhood Leukemia Study. AML’s with similar age distribution to the ALL’s were utilized as controls, to represent the infection history of an immune suppressed child. We interrogated the viral metagenomes of 60 ALL’s and 30 AML’s, pooled and reduced with ribosomal capture, then sequenced with high coverage next generation RNA sequencing on an Illumina HiSeq 2500. The resulting sequences were analyzed using a custom bioinformatics pipeline, DARK, to remove human sequences, to identify viral transcripts, and to categorize all transcripts present in the pre-treatment bone marrow. Bacterial metagenomes of the same subjects were analyzed using 16S rRNA amplicon sequencing and the QIIME pipeline.Results: We analyzed over 500 million 100bp reads and categorized human, non-human and unknown sequences. We found striking differences between the viral metagenomes of childhood ALL and AML, with ALL subjects presenting with a greater viral infection load than AML’s. Furthermore, differences in the presence of transcripts aligning to both exogenous viruses and endogenous retroviruses were observed with some viruses occurring exclusively in ALL’s. We found no difference between the bacterial metagenomes of childhood ALL and AML suggesting no differential contamination occurred during collection and analysis.Discussion: This study provides the first measurement of all identifiable infectious agents in both childhood ALL an AML. Our data suggest that bacteria do not play a role in the etiology of ALL. The greater number and frequency of viral infectious agents were observed in ALL versus AML contribute to an etiologic hypothesis of ALL involving viral infections yet require replication and additional follow up. Moreover the clinical relevance of these infections and their relation to outcome should be investigated.ReferencesGilham, C., J. Peto, J. Simpson, E. Roman, T. O. Eden, M. F. Greaves, F. E. Alexander and U. Investigators (2005). "Day care in infancy and risk of childhood acute lymphoblastic leukaemia: findings from UK case-control study." BMJ 330(7503): 1294.Greaves, M. F. (1997). "Aetiology of acute leukaemia." Lancet 349(9048): 344-349.Greaves, M. F. and F. E. Alexander (1993). "An infectious etiology for common acute lymphoblastic leukemia in childhood?" Leukemia 7(3): 349-360.Kinlen, L. J. (1995). "Epidemiological evidence for an infective basis in childhood leukaemia." Br J Cancer 71(1): 1-5.Ma, X., K. Urayama, J. Chang, J. L. Wiemels and P. A. Buffler (2009). "Infection and pediatric acute lymphoblastic leukemia." Blood Cells Mol Dis 42(2): 117-120.O’Connor, S. M. and R. S. Boneva (2007). "Infectious etiologies of childhood leukemia: plausibility and challenges to proof." Environ Health Perspect 115(1): 146-150. DisclosuresNo relevant conflicts of interest to declare.

413 Next-generation sequencing identifies the mechanism of tumourigenesis caused by loss of SMARCB1 in malignant rhabdoid tumours

significant antitumor activity in preclinical models of childhood cancers that may be prioritized for clinical testing. The PPTP has characterized a panel of 23 cell lines and 57 xenograft models in the primary testing panel representing most of the more common pediatric solid malignancies and acute lymphoblastic leukemia subtypes (ALL). Additional cell line-derived and PDX models are available for expanded testing should an agent show activity against specific cancer types in the primary screen. Molecular characterization includes expression profiles, SNP analysis, and exome sequencing for all models. Agents tested include small molecule drugs, antibodies, antibody-conjugates, and replication competent viruses. The preclinical models accurately identified known clinically active drugs (vincristine, cyclophosphamide, cisplatin, topotecan), with an overall response rate of 40% (70/175 tests). In contrast, the overall response rate for 21 signaling inhibitors was ~2%, despite clear pharmacodynamic evidence showing target inhibition in several cases. For example, the MEK inhibitor selumetinib (AZD6244) was active against only a BRAF(V600E) mutated glioma xenograft among 45 models tested. Inhibitors of the IGF-1 receptor-TOR pathway induced regressions in 9 of 321 models tested (2.8%), with activity largely limited to sarcoma. Agents showing high activity at exposures in mice not achieved in patients at comparable schedules included the Aurora A kinase inhibitor alisertib, the Polo-like kinase inhibitor BI-6727, and the pre-pro-drug PR-104. At relevant patient exposures PR104 demonstrated activity only in T cell ALL models that have high AKR1C3 activity. The tubulin binding agent eribulin demonstrated broad activity against solid tumors and ALL models at dose levels relevant to clinical exposure, including 4 of 5 Ewing sarcoma models in which vincristine had low activity. Genomic explanations for ‘exceptional responders’ were identified for the MEK inhibitor selumetinib (see above) and for the PARP inhibitor BMN 673 (PALB2 biallelic mutations). Agents demonstrating high activity against leukemia models included the CD19-targeted antibody drug conjugate SAR-3419, the MDM2 inhibitor RG7112 and the Bcl-2 family inhibitor ABT-263. Agents (or combinations) that the PPTP identified as active that have advanced to clinical testing in children include: selumetinib in low-grade glioma (NCT01089101); alisertib in ALL and neuroblastoma (NCT01154816); NTX-010 (NCT01048892); temsirolimus + cyclophosphamide + vinorelbine for rhabdomyosarcoma (NCT01222715); BMN 673 + temozolomide (NCT02116777), and eribulin (NCT02082626). Supported by NO1-CM-42216 from the NCI.

Blinatumomab, a Bi-Specific Anti-CD19/CD3 BiTE(®) Antibody for the Treatment of Acute Lymphoblastic Leukemia: Perspectives and Current Pediatric Applications.

Leukemia is the most common childhood malignancy and acute lymphoblastic leukemia (ALL) represents the largest sub-type. Despite remarkable improvements over the last 40 years, standard therapy fails in 10–20% of newly diagnosed patients. Survival for children with relapsed ALL is poor, and the development and implementation of novel therapeutic strategies in pediatric ALL are critical to further advancements. Immunotherapeutic approaches have been central to more novel ALL therapies. However, more recent innovation in antibody engineering has improved potency and efficacy, and antibody–drug conjugates (ADCs) are an especially attractive option in severely immunocompromised patients. An even more sophisticated antibody design is that of bi-specific T-cell engaging or BiTE® antibodies, which directly recruit effector T cells to augment the anti-neoplastic effect. This review focuses on blinatumomab, a bi-specific anti-CD19/CD3 antibody that has shown efficacy in adult patients with precursor B-ALL and is currently being evaluated in the pediatric setting.

Abstract 5028: Epidemiology of childhood leukemia/lymphoma in resource-poor countries: Nature's manifestation of Ludwik Gross's experiments on environmental influence on animal leukemogenesis.

Abstract Epidemiological features of childhood hematological neoplastic diseases (CHND) in resource-poor countries (RPC) that differentiate them from those of affluent countries (AC) include ∼10-fold decreased (DCR) incidence (INC) (x10−5) of common acute lymphoblastic leukemia (c-ALL) subtype, &amp;gt;5-fold increased (ICR) INC of B-ALL subtype/Burkitt's lymphoma (BL), and 20-60-fold ICR INC of chloroleukemia, as well as absence of peak ALL INC among under-5 year olds in RPC (Nigerian data) as compared to AC. To assess lifestyle influences on CHND in an African city, patients (pts) receiving care for CHND at the University College Hospital, Ibadan, Nigeria (IBN) between 9/1982 and 12/1984 underwent routine laboratory assessment and immunophenotypic cell studies, using standard international reagents and indirect fluorescent microscopy. Spatial distribution of cases and disease INC in locales of the city (LC), one of Africa's largest indigenous cities (population 1-2 million), was determined and correlated with lifestyle indices of inhabitants. LC were: central area (75%) lacking in sanitary infrastructure and inhabited by poor illiterate peasant farmers and petty traders (annual income [AI] &amp;lt;$1,000) (zone 1), surrounded by semi-developed area (zone 2) (12.5%), of semi-skilled/literate workers and traders (AI: $1500-$9000) and modern suburbs (zone 3) (12.5%) of educated professionals (AI: &amp;gt;$10,000). In zone 1, BL INC: 1.81-3.62 or &amp;gt;9-fold, AML/AMML± chloroma (CA), INC: 0.41-0.82 or &amp;gt;2-fold, and ALL INC: 0.25-0.50 or 0.3 compared to zone 2+3. BL INC: 0 in zone 3. In zone 1, time-space clustering (TSC) occurred13 times in 35 BL pts, 4 times in 9 AML/AMML±CA pts, but none in ALL. No TSC of BL or AML/AMML±CA, but 3 of ALL in 9 pts occurred in zones 2+3. Conclusion: CHND in IBN is reminiscent of the experiments of Ludwik Gross: following underfeeding of AK mice, a delay in the onset and a reduction in the rate of virus-induced leukemia occurred. Thymectomy inhibited or considerably delayed the development of lymphatic leukemia, and frequently caused the myelogenous forms to appear later in life, often in the form of chloroleukemia. At least, two other hypothetical models have been proposed to explain the pathogenesis of childhood leukemias, including “abnormal immune response to common infection(s)” (Greaves), and “population mixing” (Kinle). There are probably many causes of CHND. Given the high-risk of calorie-malnutrition in zone 1, and the associated risk of maternal and fetal malnutrition causing thymo-lymphatic deficiency, CHND pattern in zone 1 may be Nature's manifestation of the outcome of Gross's experiments, while other models may be relevant for the observation in zones 2+3 of IBN and other RPC. CHND studies in RPC with appropriate technology and reagents could unveil potentials for universal control and prevention of CHND. Citation Format: Christopher K.O. Williams. Epidemiology of childhood leukemia/lymphoma in resource-poor countries: Nature's manifestation of Ludwik Gross's experiments on environmental influence on animal leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5028. doi:10.1158/1538-7445.AM2013-5028

Philadelphia positive chronic myeloid leukemia mimicking acute lymphoblastic leukemia in a child

Chronic myeloid leukemia (CML) is a rare hematological malignancy in children. The more common acute lymphoblastic leukemia (ALL) needs to be excluded in cases with lymphoid blast crisis phase of childhood CML. A 12-year-male child with generalized lymphadenopathy and hepatosplenomegaly had a crisis phase of presentation instead of the usual chronic phase of onset of disease. Hematological tests and molecular study were performed. Peripheral blood, bone marrow and lymph node fine-needle aspiration cytology smear showed features of CML in blast crisis. Blast cells were myeloperoxidase negative. Philadelphia chromosome with major BCR-ABL fusion product and B-cell lineage immunomarkers indicated CML origin of blasts rather than de novo ALL. Usually myeloid blast crisis is common in CML but the present case had lymphoid blast crisis which is less commonly documented. We report a case of ‘adult form of CML in the juvenile age group with lymphoid blast crisis and extramedullary blast proliferation.’

Histopathological and Immunohistochemical Characterization of Spontaneously Occurring Uterine Deciduomas in Young Adult Rats

Uterine deciduomas were found in two female virgin rats, a 15-week-old Lewis rat and a 7-week-old Sprague-Dawley rat. The firm white nodules were located at the base of unilateral uterine horns and were approximately 6 mm and 4 mm in diameter. Histopathologically, the nodules were composed of three areas, each with a distinct type of proliferating cells: large epithelioid decidual cells with round nuclei, prominent nucleoli and abundant eosinophilic cytoplasm (antimesometrial region); compact spindle-shaped cells with oval nuclei and vacuolar cytoplasm (transitional region); and pleomorphic and spiny cells with round to oval nuclei and compact eosinophilic cytoplasm (mesometrial region). These cells proliferated in sheet-like arrangements and transformed into the other types of cells located in surrounding regions. Immunohistochemically, proliferating cells in all regions were strongly positive for proliferating cell nuclear antigen. The proliferating cells were positive for vimentin, and large decidual cells were positive for common acute lymphoblastic leukemia antigen 10, a marker of uterine interstitial cells. Large decidual cells were positive for α-smooth muscle actin and desmin, suggesting differentiation into muscular cells. Progesterone receptor was expressed in all cell types; however, estrogen receptor α was not expressed in the antimesometrial region. These extremely rare tumor-like nodules represent nonneoplastic lesions referred as decidual reactions of endometrial interstitial cells, and their biological behavior is that of a space-occupying benign tumor in young rats. Our cases might provide information as a historical control in toxicity and pharmacological studies in rats.

Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgm, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.

Myoepithelial cell differentiation markers in prepubertal bovine mammary gland: Effect of ovariectomy

We reported previously that ovariectomy alters prepubertal development of mammary myoepithelial cells (MC) by mechanisms that are not well understood. Therefore, in the present study, we analyzed expression of 2 myoepithelial differentiation markers, α-smooth muscle actin (SMA) and the common acute lymphoblastic leukemia antigen (CD10), in mammary parenchymal tissue from intact (INT) and ovariectomized (OVX) heifers. On d 40, Holstein heifers underwent either an ovariectomy (OVX; n=16) or a sham (INT; n=21) operation. At 55, 70, 85, 100, 130, and 160 d of age, tissues were collected, and multispectral imaging was used to quantify immunofluorescent staining for myoepithelial cell (MC) markers. Fluorescent intensity (FI) of the markers was normalized against a control sample. In the basal epithelial layer, CD10 FI was less and SMA FI was greater in OVX than INT. The ratio of SMA to CD10 FI, as a proxy indicator for MC differentiation, was greater in tissue from OVX compared with INT heifers after 55 d of age. The staining for SMA was frequently more intense along the basal aspect of cells, whereas CD10 expression was localized on the apical surface of the MC. In mammary tissue from both INT and OVX heifers, we observed basal cells that were negative for both CD10 and SMA, some of which appeared to span the distance from basement membrane to the ductal lumen. Interestingly, we also observed CD10+ cells adjacent to the ductal lumen, a situation that was more prevalent in OVX than in INT heifers. Also, ovariectomy affects MC expression of both SMA and CD10, as well as the pattern of MC development. Myoepithelial cells are known to limit parenchymal growth in other species. Involvement of MC as regulators of prepubertal bovine mammary development is worthy of further investigation.

Are cup-like blasts specific to AML patients with FLT3 ITD and a normal karyotype? An ALL case report and review of the literature.

Cup-like morphology is defined as cup-like nuclear invagination spanning ≥25% of the nuclear diameter in >10% of blasts. Studies have shown that FLT3 ITD and normal cytology are strongly associated with cup-like morphology in acute myeloid leukemia (AML) patients. Herein we describe a patient with cup-like blasts that was diagnosed and treated for common acute lymphoblastic leukemia (ALL). In contrast to the literature, the presented case was Philadelphia chromosome positive and FLT3 ITD negative.

Concise Review: Neutral Endopeptidase (CD10): A Multifaceted Environment Actor in Stem Cells, Physiological Mechanisms, and Cancer

CD10 is a remarkable member of the major class of widely expressed cell surface proteins, endopeptidases. First identified in leukemia as a tumor-specific antigen (common acute lymphoblastic leukemia antigen), CD10 has become largely used in cancer diagnosis. However, its function in oncogenesis remains unclear. We previously identified CD10 as a tool to access sphere-forming cells and showed its involvement in mammary stem cell (SC) regulation. We further illustrated that its enzymatic activity is involved, through signaling peptides, in SC maintenance. Therefore, CD10 is not only a cell surface marker in normal and malignant contexts but also affects the extracellular environment and plays a key role in regulation of a number of biological functions and likely in SC. In tumors, the "niche" favors the survival of sheltered cancer SC whose eradication has become the new challenge in oncology. This highlights the importance of understanding the role of CD10 in cancer SC. We will review the characteristics, main functions, and mechanism of action of CD10. Finally, we will review its clinical use and involvement in cancer.

Time trend in frequency of occurrence of major immunophenotypes in paediatric acute lymphoblastic leukemia cases as experienced by Cancer Institute, Chennai, south India during the period 1989-2009

Pediatric acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disease and socioeconomic and environmental factors are considered to be an important determinant of its immunophenotype. The aim of this analysis is to study the time trend in the immunophenotype of pediatric acute lymphoblastic leukemia (ALL) cases in our geographic setting. A total of 639 new pediatric ALL cases immunophenotyped during 1989-2009 forms the basis of this analysis. Representative bone marrow or peripheral blood of these patients was immunophenotyped flowcytometrically using an extensive panel of monoclonal antibodies. During early phase of our study we noticed a relative excess of T-ALL and a paucity of common acute lymphoblastic leukemia (C-ALL) in contrast to western data. Over a period of 20 years we witnessed a gradual reduction in pediatric T-ALL cases and a proportionate increase in C-ALL cases. We find that this change of pattern is synchronizing with the socioeconomic and industrial development prevailing in our geographic setting and suggest a possible link between the predominant immunophenotype of pediatric ALL cases and the environmental and socioeconomic factors prevailing in that locality.

Paraneoplastic Myopathy as a Very Rare Manifestation of Acute Lymphoblastic Leukemia

We present a 9-year-old girl who developed acute muscular weakness of proximal muscles of the upper and lower limbs. Investigations revealed a common acute lymphoblastic leukemia. The neuromuscular symptoms are classified as a paraneoplastic neurological syndrome (PNS). Under chemotherapy according to ALL-BFM-2000 protocol symptoms resolved within 4 weeks. This case presents a rare manifestation of acute lymphoblastic manifestation.